生物钟蛋白REV-ERB激动剂对人胰腺癌细胞PANC-1的杀伤作用及机制研究  

Antitumor effect and mechanisms of circadian protein REV-ERB agonist on human pancreatic cancer PANC-1 cells in vitro

作　　者：付祥 许重远[1,2] FU Xiang;XU Zhong-yuan(Clinical Pharmacy Center,Nanfang Hospital,Southern Medical University,Guangzhou 510515,Guangdong Province,China;National Medical Products Administration Key Laboratory for Research and Evaluation of Drug Metabolism,Southern Medical University,Guangzhou 510515,Guangdong Province,China)

机构地区：[1]南方医科大学南方医院临床药学中心,广东广州510515 [2]南方医科大学药物代谢研究与评价重点实验室,广东广州510515

出　　处：《中国临床药理学杂志》2022年第22期2684-2687,共4页The Chinese Journal of Clinical Pharmacology

基　　金：国家科技重大专项重大新药创制基金资助项目(2020ZX09201-017);深圳市医药物资储备目录体系建设研究基金资助项目(2020A00042)。

摘　　要：目的研究生物钟蛋白REV-ERB激动剂SR9009对人胰腺癌细胞PANC-1的杀伤作用及其潜在机制。方法将处于对数生长期的人胰腺癌PANC-1细胞,分别用0,6.25×10^(-1),1.25,2.5,5,10,20μmol·L^(-1)REV-ERB激动剂SR9009处理。处理72 h后以细胞计数法-8(CCK-8)法检测人胰腺癌细胞的活力;用流式细胞术分析细胞凋亡的情况;用蛋白质印迹法检测不同浓度的SR9009作用于PANC-1细胞后细胞凋亡和自噬相关蛋白的表达情况。SR9009作用于PANC-1细胞时,分为加泛胱天蛋白酶(caspase)抑制剂z-VAD-fmk组和不加z-VAD-fmk组,通过CCK-8法检测PANC-1细胞活力的变化。结果0,6.25×10^(-1),1.25,2.5,5,10,20μmol·L^(-1) SR9009组作用于PANC-1细胞72 h后,PANC-1的细胞活力分别为(100.02±4.79)%,(100.26±2.18)%,(79.12±2.64)%,(61.44±1.98)%,(55.34±2.30)%,(28.05±1.56)%,(22.31±1.94)%,细胞活力下降呈明显浓度依赖;流式细胞术说明人胰腺癌细胞凋亡率增加;蛋白质印迹实验表明caspase 3剪切蛋白的表达随着作用浓度升高而上调,自噬相关蛋白5(ATG5)随着SR9009作用浓度的升高而降低,自噬相关蛋白P62随着SR9009作用浓度的升高而表达增加。CCK-8实验表明加入z-VAD-fmk后,SR9009诱导的细胞毒性被削弱。结论生物钟蛋白REV-ERB激动剂对人胰腺癌细胞PANC-1有显著的抗肿瘤作用,这种作用可能是通过诱导caspase依赖的细胞凋亡和抑制细胞自噬实现的。Objective To explore the cytotoxicity effect and mechanisms of circadian protein REV-ERB agonist on human pancreatic cancer cells in vitro.Methods The human pancreatic cancer cells in the logarithmic phase of growth were treated with 0,6.25×10^(-1),1.25,2.5,5,10,20μmol·L^(-1) REV-ERB agonist SR9009.Cell viability was detected by cell counting kit-8(CCK-8)assay after 72 h.The apoptosis rates of pancreatic cells were tested by the flow cytometry.Western blotting was used to analyze the protein expression levels of cell apoptosis and autophagy after treatment of SR9009.The PANC-1 cells were incubated with the indicated concentration of SR9009 in the presence or absence of z-VAD-fmk for 72 h,then CCK-8 assay was applied to measure the cytotoxicity.Results The cell viability of 0,6.25×10^(-1),1.25,2.5,5,10,20μmol·L^(-1) experimental groups were(100.02±4.79)%,(100.26±2.18)%,(79.12±2.64)%,(61.44±1.98)%,(55.34±2.30)%,(28.05±1.56)%,(22.31±1.94)%,which were significantly reduced in PANC-1 cells in a dose-dependent way after treatment of SR9009 for 72 h.The flow cytometry demonstrated that the apoptosis rate of pancreatic cancer cells significantly increased.Western blotting analysis indicated that the protein level of cleaved-caspase 3 up-regulated as the concentration increased,and autophagy related protein autophagy-related protein 5(ATG5)down-regulated while P62 increased.CCK-8 assay demonstrated that SR9009-induced cytotoxicity could be partially restored by z-VAD-fmk.Conclusion Circadian protein REV-ERB agonist SR9009 could induced significant antitumor effect on human pancreatic cancer PANC-1 cells in vitro,which may be owing to cell apoptosis and the inhibition of autophagy.

关 键 词：人胰腺癌 凋亡 自噬 

分 类 号：R97[医药卫生—药品]